Estrogen Improves Insulin Sensitivity and Suppresses Gluconeogenesis Via the Transcription Factor Foxo1
Total Page：16
File Type：pdf, Size：1020Kb
Diabetes Volume 68, February 2019 291 Estrogen Improves Insulin Sensitivity and Suppresses Gluconeogenesis via the Transcription Factor Foxo1 Hui Yan,1 Wangbao Yang,1 Fenghua Zhou,1 Xiaopeng Li,1 Quan Pan,1 Zheng Shen,1 Guichun Han,2 Annie Newell-Fugate,2 Yanan Tian,2 Ravikumar Majeti,3 Wenshe Liu,4 Yong Xu,5 Chaodong Wu,1 Kimberly Allred,1 Clinton Allred,1 Yuxiang Sun,1 and Shaodong Guo1 Diabetes 2019;68:291–304 | https://doi.org/10.2337/db18-0638 Premenopausal women exhibit enhanced insulin sensi- be a potential approach to modulate glucose metabolism tivity and reduced incidence of type 2 diabetes (T2D) and prevent diabetes. compared with age-matched men, but this advantage disappears after menopause with disrupted glucose ho- meostasis, in part owing to a reduction in circulating The prevalence of type 2 diabetes (T2D) has shown sex b 17 -estradiol (E2). Fasting hyperglycemia is a hallmark of disparities, with a reduced incidence in women (1). Both T2D derived largely from dysregulation of hepatic glu- clinical and animal studies show a strong correlation be- cose production (HGP), in which Foxo1 plays a central tween estrogen deficiency and metabolic dysfunction (2,3). METABOLISM role in the regulation of gluconeogenesis. Here, we in- The reduction of estrogen in postmenopausal women vestigated the action of E2 on glucose homeostasis in accelerates the development of insulin resistance and male and ovariectomized (OVX) female control and liver- T2D (4). Clinical trials of estrogen replacement therapy fi speci c Foxo1 knockout (L-F1KO) mice and sought to in postmenopausal women demonstrated an amelioration understand the mechanism by which E2 regulates glu- of insulin resistance and reductions of plasma glucose level coneogenesis via an interaction with hepatic Foxo1. In and incidence of T2D (5,6). However, the potential risk of both male and OVX female control mice, subcutaneous breast cancer and stroke upon estrogen therapy under- E implant improved insulin sensitivity and suppressed 2 scores 17b-estradiol (E ) as a therapeutic agent (7,8). gluconeogenesis; however, these effects of E were 2 2 Thus, understanding of tissue-specificE action and its abolished in L-F1KO mice of both sexes. In our use of 2 molecular mechanism in metabolic regulation is required mouse primary hepatocytes, E2 suppressed HGP and gluconeogenesis in hepatocytes from control mice but for the development of targeted estrogen mimics convey- fi failed in hepatocytes from L-F1KO mice, suggesting that ing metabolic bene t without side effects. The maintenance of glucose homeostasis depends on Foxo1 is required for E2 action on the suppression of glucose uptake in muscle and adipose tissue and glucose gluconeogenesis. We further demonstrated that E2 sup- presses hepatic gluconeogenesis through activation of production through gluconeogenesis and glycogenolysis in estrogen receptor (ER)a–phosphoinositide 3-kinase– liver (9). Estrogen reduces glucose level, but it is thought Akt–Foxo1 signaling, which can be independent of insulin to be derived from the promotion of glucose uptake in receptor substrates 1 and 2 (Irs1 and Irs2), revealing an muscle (10). However, some studies have also indicated important mechanism for E2 in the regulation of glucose that estrogen suppresses glucose production in the liver homeostasis. These results may help explain why pre- (11–13) and that impairing the estrogen receptor (ER)- menopausal women have lower incidence of T2D than mediated signaling results in hepatic insulin resistance and age-matched men and suggest that targeting ERa can hyperglycemia with elevated hepatic glucose production 1Department of Nutrition and Food Science, College of Agriculture and Life Corresponding author: Shaodong Guo, [email protected] Sciences, Texas A&M University, College Station, TX Received 14 June 2018 and accepted 3 November 2018 2Department of Physiology and Pharmacology, College of Veterinary Medicine and © 2018 by the American Diabetes Association. Readers may use this article as Biomedical Sciences, Texas A&M University, College Station, TX long as the work is properly cited, the use is educational and not for profit, and the 3Department of Pharmaceutical Sciences, College of Pharmacy, Texas A&M work is not altered. More information is available at http://www.diabetesjournals University, College Station, TX .org/content/license. 4Department of Chemistry, Texas A&M University, College Station, TX 5Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 292 Estrogen, Foxo1, and Glucose Homeostasis Diabetes Volume 68, February 2019 (HGP) (14,15). These lines of evidence underscore the im- determination of serum insulin, glucagon, and other hor- portance of estrogen signaling in the liver in control of mone levels by Bio-Plex mouse diabetes immunoassay glucose homeostasis; yet, the liver-specific action of estro- (Bio-Rad, Hercules, CA) and serum E2 concentration by gen on HGP and its mechanism remain unclear. an ELISA kit (Cayman, Ann Arbor, MI). Liver glycogen Foxo1, a member of O-class forkhead transcription content was measured from mice fasted 16 h as previously factor, is a predominant regulator for HGP, promoting described (24). gene transcription of the rate-limiting gluconeogenic en- zyme glucose-6-phosphatase (G6pc) (16,17). Foxo1 is sup- Cell Cultures and HGP Assay pressed by insulin through the activation of Akt via the Primary hepatocytes were isolated from 8- to 16-week-old insulin receptor substrate (Irs)1 and Irs2-associated phos- mice and cultured in DMEM with 10% FBS as previously phoinositide 3-kinase (PI3K) (18,19). In a T2D mouse described (21). Cells were serum starved for 6 h before model, insulin fails to activate Akt and suppress Foxo1, treatment with 100 nmol/L E2 and a variety of signal trans- resulting in enhanced HGP and hyperglycemia; this can duction inhibitors, which were applied 30 min prior to E2.For be prevented by deletion of hepatic Foxo1 (20). In this HGP assay, freshly isolated hepatocytes were cultured in study, we investigated the effect of E2 on the maintenance DMEM with 2% FBS. After 3 h of attachment, cells was of glucose homeostasis and the involvement of hepatic cultured in HGP buffer (120 mmol/L NaCl, 5.0 mmol/L KCl, Foxo1 in mice of both sexes. We further examined E2 2.0 mmol/L CaCl2, 25 mmol/L NaHCO3, 2.5 mmol/L regulatory mechanisms for HGP and gluconeogenesis in KH2PO4, 2.5 mmol/L MgSO4,10mmol/LHEPES,0.5% primary hepatocytes. In particular, we tested the hypoth- BSA, 10 mmol/L sodium DL-lactate, and 5 mmol/L pyruvate, esis that hepatic Foxo1 plays important roles in the E2 pH 7.4) and treated with chemicals. Culture medium was action of suppressing hepatic gluconeogenesis through collected to determine glucose production using an Amplex ER-associated Akt signaling. Red Glucose Assay kit (Invitrogen, Carlsbad, CA). Total HGP, glycogenolysis, and gluconeogenesis were determined and calculated as previously described (25). RESEARCH DESIGN AND METHODS Animals Quantitative Real-time PCR Animal protocols were approved by the institutional an- RNA was extracted with Trizol reagent (Invitrogen) and imal care and use committee at Texas A&M University. used for cDNA synthesis via an iScript cDNA Synthesis kit Liver-specific Foxo1 knockout (L-F1KO) mice were gener- (Bio-Rad). Gene expression was measured with SYBR L/L ated by breeding floxed Foxo1 mice with albumin-Cre Green Supermix in real-time PCR (Bio-Rad) using primers mice as previously described (21). The Cre recombinase as previously described and cyclophilin gene as an internal induced by the mouse albumin enhancer/promoter shows control (21). no influence on animal performance, as previously reported, and either the floxed-Foxo1L/L or albumin-Cre littermates Protein Immunoblotting were used as control mice in our studies (22,23). Eight- to An equal amount of protein was resolved in SDS-PAGE and 2 12-week-old control (Foxo1L/L) and L-F1KO (albumin-Cre+/ :: transferred to nitrocellulose membrane for Western blot. Foxo1L/L) mice on a mixed genetic background of C57BL/6J Antibodies for Foxo1, phosphorylated Foxo1 at Ser253, and 129/Sv were fed a standard chow diet (54% calories Akt, phosphorylated Akt at Ser473, Pck1, and GAPDH from carbohydrate, 14% from fat, and 32% from protein were purchased from Cell Signaling Technology. Protein and 3.0 kcal/g) (Envigo Teklad Diet) ad libitum. densitometry was performed and analyzed using ImageJ as previously described (21). Ovariectomy and E2 Replacement Control and L-F1KO mice were randomly assigned to Statistical Analyses experimental groups (n =4–7). All mice were subjected Data were analyzed by one-way or two-way ANOVA to fi to the subcutaneous implantation of placebo or E2 pellet determine the signi cance of the model as appropriate. (0.05 mg/pellet, 60-day release) (Innovative Research of The interaction between E2 and L-F1KO was analyzed in America, Sarasota, FL). Female mice underwent a bilateral two-way ANOVA to determine the dependency. Differ- ovariectomy (OVX) surgery (except for intact control mice) ences between groups were determined by Tukey post t P , at the time of pellet implantation. hoc test or Student test as appropriate. 0.05 was considered indicative of statistical significance. Results are 6 Metabolic Analyses presented as means SEM. Blood glucose levels were measured using a glucometer (Bayer, Whippany, NJ). Glucose tolerance tests (GTTs) RESULTS and pyruvate tolerance tests (PTTs) were performed in E2 Regulates Glucose Homeostasis Depending on mice fasted for 16 h. Insulin tolerance tests (ITTs) were Hepatic Foxo1 in Male Mice conductedinmicefastedfor5h.Serumsampleswere We determined the role of estrogen in control of glu- collected from mice fasted for 16 h by cardiac puncture for cose homeostasis and its dependence on hepatic Foxo1. diabetes.diabetesjournals.org Yan and Associates 293 Generally, intact female mice exhibited a 16% lower fasting glucose compared with intact females (intact 51 6 glucose level compared with male mice (intact female 51 6 2.8 mg/dL vs.